Prestin, a motor protein of cochlear outer hair cells, is the basis for cochlear amplification. Prestin belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Individual members of this 11-member family serve two fundamentally distinct functions. While all prestin paralogs (i.e., all other members in this family) transport different anion substrates across a variety of epithelia, prestin (SLC26A5) is the only member serving as a molecular motor. The goal of the proposed research is to investigate the molecular mechanisms of how prestin works. We have identified two regions (residues 158-168 and residues 256-276) that are well conserved among mammalian species but highly variable among prestin orthologs and SLC26A paralogs. Our overall hypothesis is that these amino acid sequences represent the essential motif for motor function. This project has three specific aims: Aim 1 will determine whether residues 158-168, located in the external loop 2 of the prestin molecule, represent the essential motif for motor function. Aim 2 will determine whether the length and constitution of the 11-amino acid sequence are critical for voltage-sensing/motor function. Aim 3 will determine whether residues 256-276, located in the transmembrane domain 6 of the prestin molecule, are important for enhancing motor function. Site-directed mutagenesis will be used to swap these regions between gerbil prestin and human pendrin. Pendrin (SLC26A4), one of the paralogs, is a sodium-independent chloride/iodide transporter. Mutations in this gene are associated with Pendred syndrome, the second most common form of syndromic deafness. Nonlinear capacitance and somatic motility, two hallmarks of the motor function of prestin, will be measured from chimeric pendrin-transfected HEK cells, using the voltage-clamp technique and a photodiode-based displacement measurement system. A gain of motor function is expected in the chimeric pendrin. A radioisotope uptake technique will be used to determine whether the chimeric pendrin retains its transport function. The proposed experiments are significant for the fundamental understanding of how prestin and pendrin work.